Method and product for the treatment of gastric disease

ABSTRACT

This invention describes a product obtained from the isolation and concentration of specific immunoglobulins (antibodies) derived from the mammary secretions of cows immunized with Helicobacter pylori. The product is useful in preparing formulations for the treatment and/or prevention of gastric diseases.

This is a continuation of application Ser. No. 07/559,793, filed Jul.30, 1990, now abandoned.

TECHNICAL FIELD

This invention relates to the isolation of specific immunoglobulinswhich, in the preferred embodiment, are isolated from the colostrum ormilk of cows immunized with Helicobacter pylori. This invention is alsodirected to a method of use of these specific antibodies in thepreparation of novel formulations useful in the enteral treatment ofgastric disease.

BACKGROUND

The present invention relates to a process for the production of aprotein concentrate containing immunological factors preferably oflactic origin and to a specific immunoglobulin population. Moreparticularly, this invention relates to active immunoglobulinsexhibiting specificity against the microorganism, Helicobacter pylori(formerly referred to in the literature as Campylobacter pylori) and theuse of this protein in the management of Helicobacter pyloricolonization in the gastrointestinal (GI) tract. More specifically, thepresent invention relates to the injection of lactating mammals withHelicobacter pylori, subsequent isolation and concentration ofantibodies from the colostrum (or milk) produced by the immunizedmammals and use of this concentrate, by way of enteral ingestion, inreducing the infectivity of Helicobacter pylori resident in thegastrointestinal tract. This protein (immunoglobulin) concentrate isuseful in the treatment of pathological sequela associated withHelicobacter pylori colonization of the GI tract including gastritis andpeptic ulcer disease.

In general, the prior art discloses the introduction and use ofimmunological factors of lactic origin into-dietetic products fornewborn babies and infants. The oral ingestion of these dieteticproducts being intended to enable these immunological factors to beutilized in the development of protection against the consequences ofmicrobial infection within the GI tract.

U.S. Pat. Nos. 3,992,521 and 3,984,539 disclose a process for obtainingan immune product containing antibodies from the serum of a horse or cowand the immunoglobulin product itself. These patents do not suggest nordisclose the specific immunoglobulin of the present invention, nor itsmethod of production and isolation, nor its intended method of use forthe treatment of Helicobacter pylori induced gastritis.

U.S. Pat. No. 3,123,230 discloses a method for producing antibodieswhich consists of injecting a lactating mammal with a mixture of killedmicroorganisms and isolating the antibodies from serum or milk. Thispatent does not suggest nor disclose that Helicobacter pylori can inducean antibody response nor the specific method of treatment employed inthis invention.

British Patent No. 1,573,995 discloses and claims a process for theproduction and isolation of immunoglobulins exhibiting specificityagainst Escherichia coli. This patent does not suggest thatmicroorganisms other than E. coli are useful. In similar fashion, thefollowing references disclose the same type of process: 1) H. Hilpert,et al., Proceedings of the 13th Symposium Swedish NutritionalFoundation; and 2) C. Mietens, et al., European J. Pediatrics, 132,239-252, (1979).

Ebina and colleagues disclose the immunization of cows with humanrotavirus and the isolation of immunoglobulin to the virus from the milkof cows. This immunoglobulin was orally administered to children and wasfound to reduce the frequency of the outbreak of diarrhea. See Ebina, etal., The Lancet (Oct. 29, 1983), 1029-1030, (1983); and Ebina, et al.,Med. Microbiol. Immunol., 174, 177-185, (1985). These references do notsuggest nor disclose that immunoglobulins to Helicobacter pylori wouldbe useful in the management Helicobacter pylori induced gastritis.

U.S. Pat. No. 4,051,235 discloses the isolation of immunoglobulins fromthe milk of vaccinated cows by coagulating the milk, recovering thelactoserum (whey) and selectively precipitating the immunoglobulins withammonium sulfate, followed by dialysis against water, filtration anddrying. Seroprotection tests demonstrated that the protein concentratesof U.S. Pat. No. 4,051,231 provided local passive immunity in theintestine without resorption and without any significant loss ofactivity in the digestive tract, thereby providing generalized passiveprotection against certain enteropathogenic bacteria and/or viruses.This patent does not suggest nor disclose that such antibodies couldserve to modify the course of Helicobacter pylori induced gastritis.

Much has been published regarding Helicobacter pylori itself.Helicobacter pylori is approximately 0.85 um in diameter with an averagelength of 2.9 um. The microorganism has a smooth coat and four to sixpolar flagella which are sheathed and have bulbous ends. In freshcultures this organism appears as a slender, curved Gram-negative rod.Helicobacter pylori is readily distinguished from other gastric bacteriaand spirochaetes by the absence of axial filaments in its flagella.Furthermore, optimum growth conditions for Helicobacter pylori areunusual and help to set it apart from other enteropathogens. Forexample, Helicobacter pylori requires a microaerophilic gas environment(i.e. low oxygen content) to sustain growth. Helicobacter pylori appearsto tolerate a wide range of local pH conditions and is relativelyresistant to acid conditions. It is believed that this resistance is duein part to the organism's outer protein structure which contains ureasein large amounts resulting in the cleavage of urea naturally present ingastric fluid and hence, the formation of a buffering ammonia layerimmediately around the organism.

Although a number of spiral bacteria inhabit the mouth and lowerintestinal tract of all mammals, what distinguishes Helicobacter pyloriis the is observation that it is localized almost exclusively to theluminal mucosal surface of the stomach and duodenum and generally isfound deep within the gastric pits.

It is the combination of the unusual growth requirements and intestinallocation which makes eradication and treatment of Helicobacter pylori sodifficult. The ideal antimicrobial drug suitable for the successfultreatment of Helicobacter pylori associated gastritis should exhibitlocal activity, be stable at low pH values and should be able to readilypenetrate the gastric mucosa. These desirable properties of anantimicrobial are not easily accomplished and thus, satisfactorytreatment of Helicobacter pylori with antimicrobials has yet to beaccomplished.

The development of an agent which is effective in the management ofHelicobacter pylori induced gastritis would fulfill a long felt need.

There is an emerging consensus in the field of gastroenterology thatHelicobacter pylori is a major contributing-factor in the development ofgastritis and peptic ulcer disease. Specifically, the followingreference is useful in establishing the background of the presentinvention: Campylobacter pylori, E. A. J. Rauws and G. N. J. Tytgat,editors, Adis Press Intntl. (1989).

In general, this reference discloses, at pages 138-139, the role ofHelicobacter pylori in the development of gastritis and peptic ulcerdisease. The key evidence in support of Helicobacter pylori etiology inthese conditions is based on the observation at pages 89-103, thatelimination of Helicobacter pylori from the stomach through the use ofantibiotics and/or bismuth compounds leads to a remission of the gastricdisease.

Presently, the main therapies employed in the treatment of chronicactive gastritis and peptic ulcer disease include the histamineH2-receptor antagonist's, bismuth compounds, and antibiotics. However,it is generally accepted that all currently used treatment modalititesare clinically inadequate since post-treatment relapse rates remainunacceptably high. In addition, several of these therapies areaccompanied by significant side effects. For example, effectiveantibiotic treatment of Helicobacter pylori infections requirestreatment over an extended duration (4-6 weeks) and results in theinduction of diarrhea and intestinal discomfort. The bismuth compoundsare also known to have a number of significant undesirable side effects.

To date, the preferred treatment has been dominated by the use ofH2-antagonists which result in the suppression of acid and pepsinsecretion; however, post treatment relapse rates are extremely high.Since symptoniatic relief and ulcer healing are the primary aim oftreatment, without indefinite maintenance therapy, it is becomingincreasingly apparent that a mucosal "protective agent" havingantimicrobial activity against Helicobacter pylori, is desirable.

Thus, the medical community has a need for a protective agent which canbe readily utilized in pharmaceutical and/or nutritional formulations.The present invention fulfills that need through the discovery thatenteral ingestion of immunoglobulins derived from lactating mammalsimmunized with Helicobacter pylori provides such protection.

The prior art fails to suggest, disclose or contemplate the instantdiscovery which is, in part, the use of antibodies (immunoglobulin) inthe treatment of Helicobacter pylori infection of the gastric mucosa andto the antibodies themselves.

Lactile secretion derived antibodies obtained from cows immunized withHelicobacter pylori will provide numerous advantages over other methodsof immunoglobulin production. The advantages include quantity, ease andreproducibility of immunoglobulin isolation, ease of product preparationand significant cost savings as compared to antibody and productpreparation based on other isolation methods.

One aspect of the present invention relates to a method for producing amilk based product having high immunological specific activity againstHelicobacter pylori.

Another further aspect of this invention is the specific immunoglobulinitself which is produced according to the disclosed method.

A further aspect of the present invention relates to a method fortreating mammals in order to produce milk having immunologicalcomponents which provide protection against Helicobacter pylori tosubjects imbibing same.

A further aspect of this invention is the use of these specificantibodies (immunoglobulins) in the treatment of Helicobacter pyloriinduced gastritis.

DISCLOSURE OF THE INVENTION

There is disclosed a composition of matter consisting of non-denaturedimmunoglobulins which exhibit specific activity to the bacteriumHelicobacter pylori. More specifically, an immunoglobulin isolated fromthe mammary secretions of mammals exhibiting specific activity towardsHelicobacter pylori .

There is also disclosed a medicament for gastritis caused byHelicobacter pylori which comprises non-denatured immunoglobulin whichexhibits specificity towards Helicobacter pylori. The disclosedmedicament may be used alone or in combination with a pharmacologicallyand/or nutritionally acceptable carrier and may be in a powdered orliquid form.

There is further disclosed a method for treating an individual sufferingfrom Helicobacter pylori induced gastritis, peptic ulcer disease orother diseases said method consisting of administration to theindividual in need of treatment an effective amount of a compositionwhich contains at least the non-denatured immunoglobulins havingspecificity against Helicobacter pylor.

There is also disclosed a method for producing immunoglobulinsexhibiting specificity for Helicobacter pylori which comprises the stepsof 1) immunizing a lactating or pregnant mammal with a cell suspensionof Helicobacter pylori emulsified in an adjuvant; 2) obtaining thecolostrum or milk from the mammal; and 3) isolating the immunoglobulinsfrom the secretion.

In general, the composition of matter of this invention is derived by aprocess which comprises the isolation of immunoglobulins from themammary secretions of mammals immunized with Helicobacter pylori, saidimmunoglobulins exhibiting specific antimicrobial activity againstHelicobacter pylori.

The method for the treatment of Helicobacter pylori infections,comprises the oral ingestion of an effective amount of Helicobacterpylori-specific immunoglobulins by a patient in need of treatment, saidimmunoglobulins being derived from the mammary secretions of mammalsimmunized with Helicobacter pylori. The immunoglobulins may be ingestedalone or in combination with other materials such as fats, oils andproteins.

In its broadest aspect the present invention is directed to novelcompositions which demonstrate antimicrobial activity againstHelicobacter pylori.

The novel composition of matter of this invention consists of theimmunoglobulins isolated from the mammary secretions of mammalsimmunized with Helicobacter pylori which may be utilized alone orcombined with other natural or synthetic edible products such as lipids,proteins or oils. Said composition of matter is readily employed aloneor in combination with other edible products to yield admixtures whichare useful in the treatment of gastric diseases.

Other aspects and advantages of the invention will be apparent uponconsideration of the following detailed description of the illustrativeembodiments hereof.

Best Mode

The utility of this invention was demonstrated by the ingestion of thespecific antibody (immunoglobulins) of this invention by Helicobacterpylori infected germ free piglets. Ingestion of a nutritional containingthe specific antibody (immunoglobulins) of this invention provided thereduction or elimination of Helicobacter pylori induced pathology aswell as a reduction in Helicobacter pylori bacterium colonization levelsin various gastric epithelium regions, as determined by both agar plateculture reisolation and by histologic methods.

Specific antibody to Helicobacter pylori was raised in cows (asdescribed in detail below) and characterized by standard immunochemicaltechniques as described below. Additional characterization of theantibodies can be achieved, for example, through the assessment of theirability to agglutinate Helicobacter pylori, their ability to fixcomplement in the presence of the Helicobacter pylori bacteria, theability of the antibodies to inhibit bacterial replication and theirability to specifically bind the Helicobacter pylori bacterial antigensas detected by common immunochemical methods such as immunofluorescenceand the like.

Following immunochemical characterization, the Helicobacter pylorispecific antibodies were fed to Helicobacter pylori monoinfectedgnotobiotic piglets according to the feeding regimen described below.Following feeding of Helicobacter pylori specific antibody, bloodsamples were drawn and the animals sacrificed for subsequentmicrobiological and histopathological assessment of the treatmentprotocol. The results of these studies were compared to Helicobacterpylori monoinfected gnotobiotic piglet littermates fed a nonimmune milkbased on the nutritional product, Similac® (infant nutritional productof Ross Laboratories, Division of Abbott Laboratories, Columbus, Oh.)which does not contain specific Helicobacter pylori antibodies.

As a result of these experiments, the inventors have discovered thatenteral ingestion of an the immunoglobulin product containing specificantibodies to Helicobacter pylori results in the reduction in the levelsof viable Helicobacter pylori contained within various regions of thestomach and as such provides a realistic approach for the treatment ofHelicobacter pylori induced gastritis. The Helicobacter pylori specificantibodies may be employed alone (i.e. in a liquid, tablet or capsuleform) or in combination with other pharmaceutically acceptable carrierssuch as various lipids, is proteins or oils which may al so provideadditional nutritional and/or pharmaceutical benefits.

EXPERIMENTAL

The following examples relate to the production and use of specificantibodies to Helicobacter pylori and the physiological results of suchusage. More specifically Examples 1 and 2 relate to the production andcharacterization of the immunoglobulin material isolated from pregnantcows immunized with Helicobacter pylori bacteria. Table 1 sets forth theimmunological characterization of the colostrum whey products isolatedfrom cows immunized with Helicobacter pylori as compared tonon-immunized (i.e. control) cows. This data indicates that theconcentration of Helicobacter pylori specific antibody (immunoglobulins)levels contained in whey provided by Helicobacter pylori immunized cowsincreased by over one hundred fold as compared to non-immunized cows.Example 3 relates to the biophysical and biological characterization ofthe specific immunoglobulins isolated from immunized versusnon-immunized cows and exhibiting activity specifically againstHelicobacter pylori. Tables 2 and 3 summarize the biological andbiophysical data respectively.

Examples 4, 5 and 6 relate to the formulation and use of this immunematerial in the feeding of gnotobiotic pigs preinfected withHelicobacter pylori and thus, of the utility of this material in thetreatment of Helicobacter pylori induced gastritis. The data containedin Table 4 indicates clearly that animals exposed to Helicobacter pylorithrough oral ingestion as described in Example 5 develop systemic (seracontained) antibodies to Helicobacter pylori, thereby confirming theeffectiveness of oral treatment with Helicobacter pylori as a means ofachieving Helicobacter pylori infection.

Example 7 relates to the assessment of the effect of feeding this immunematerial on the levels of viable Helicobacter pylori bacteria which cansubsequently be recovered from various gastric epithelial sites ofpiglets preinfected with Helicobacter pylori. Tables 5 and 6 summarizethese results. The data indicate markedly reduced recoveries of viableHelicobacter pylori from all gastric regions examined for animals fedthe immune product as compared to animals which received the nonimmunenutrient only. These results clearly indicate the effectiveness of usingHelicobacter pylori specific antibodies (immunoglobulins) in thetreatment of Helicobacter pylori induced gastritis.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Preparation ofAntibodies and Control Products

Whey containing Helicobacter pylori specific antibodies was preparedfrom colostrum derived from a cow immunized while pregnant with wholeformalin killed Helicobacter pylori bacteria (ATTCC Strain 26695). Thebacteria, emulsified in incomplete Freund's adjuvant, were employed at aconcentration of 5×10⁹ colony forming units (CFU)/mL. Each innoculationconsisted of 12 mL of this material. The following immunization schedulewas employed for each cow. Initially a subcutaneous (SQ) innoculation 14days prior to drying off (D-14) was given. This was followed by anintramammary booster given seven days post drying off (D+7) and a secondSQ booster given at D+30. This and similar immunization schedules aretaught by the prior art and while the above schedule fully describes themethod used, this description is not meant to limit the method ofimmunization under which the antibodies (immunoglobulins) toHelicobacter pylori can be raised since those skilled in the art willrecognize and understand that other immunization methods would givesimilar results.

Upon the birth of the calf of the immunized cow, colostrum wascollected, rennet whey prepared and the whey stored frozen at -20° C.until use. The isolation scheme for obtaining the whey is largely asdescribed in U.S. Pat. No. 4,051,235 which is herein incorporated byreference. The basic steps involved are:

Collection and freezing of the bovine colostrum. Thereafter fat isremoved from the colostrum. This is achieved by partially thawing thefrozen colostrum and removing the upper liquid portion. The remainingmaterial is then completely thawed and centrifugally separated to removeas much of the remaining fat as possible.

The defatted colostrum was precipitated by adding 1.5 mg of calciumchloride per liter of colostrum and by adding 1.5 gram of commerciallyavailable rennin per liter of colostrum. The mixture was then thoroughlystirred at 20° C. Thereafter, the solution was permitted to stand for2-5 hours while the casein in the solution precipitated. Theprecipitated casein was then removed by filtration. The resultingsolution is hereinafter referred to as "bovine colostrum whey" (BCW).The solution was then clarified by filtration and the protein andimmunoglobulin concentration were then determined using methodsdescribed below.

EXAMPLE 2 Characterization of Antibody Test Material

The bovine colostrum whey (BCW) samples were analyzed for total protein,immunoglobulin isotype type characterization, IgGl content and forspecific anti-Helicobacter pylori antibodies. The techniques used forthese assays are standard procedures employed in the art and were,respectively, dye binding methods (BioRad), radial immunodiffusion, (ICNBiochemicals), and the enzyme linked immunoassay (ELISA).

The ELISA plates were coated with a Helicobacter pylori cell lysate at3.2 ug/ml. The detecting antibody employed was a conjugate of alkalinephosphatase-coupled to a monoclonal antibody having immunospecificityfor bovine IgGl type antibodies. The indicator substrate for the assaywas p-nitrophenylphosphate. The extent of color development was measuredon a Dynatech ELISA plate reader at a visible wavelength of 490 nm andthe data analyzed according to standard statistical methods. The resultsfor these studies are summarized in Table 1. The data illustratesclearly that oral exposure to Helicobacter pylori results in over a onehundred fold increase in immunoglobulin (IgGl) concentration incolostral whey as compared to whey obtained from non-immunized cows.

                                      TABLE 1                                     __________________________________________________________________________    CHARACTERIZATION OF BOVINE COLOSTRAL WHEY PREPARATION                                                    ELISA.sup.b                                        COLOSTRAL WHEY                                                                            TOTAL PROTEIN                                                                           mg IgGl/      FOLD DIFFERENCE                           PREPARATION.sup.a                                                                         (mg/mL)   mL   TITER/mg IgGl.sup.b                                                                    FROM NON-IMMUNE                           __________________________________________________________________________    IMMUNE      176.2     125.2                                                                              615      123                                       CONTROL     Not Determined                                                                          171.2                                                                              5        1                                         (Non-Immunized)                                                               __________________________________________________________________________     .sup.a Colostral whey obtained from Helicobacter pylori immunized cow and     from the nonHelicobacter pylori immunized cow (CONTROL).                      .sup.b Nonadjusted ELISA titers on undiluted colostral whey [IMMUNE =         77,000 and CONTROL = 860]. This assay detects antibodies specifically         against Helicobacter pylori.                                             

EXAMPLE 3 Characterization of Helicobacter pylori Antibodies

Specific Helicobacter pylori antibodies may be characterized in a numberof ways. For the purpose of the present study the biological activity ofthe immunoglobulins was determined by means of an agglutination assay(Table 2) and the biophysical characterization is provided by aconsideration of the physical properties of the dominant immunoglobulinisotype contained in BCW (Table 3).

The bacterial agglutination test is a classical procedure used todetermine the presence and relative concentration of specific antibodyin sera to specific bacteria (e.g. Widal test for typhoid fever).Agglutination involves the aggregation of bacteria into large clumps asa result of the binding of specific antibody to particular sites on theouter surface antigens of the bacterium. Thus, bacterial clumping(agglutination) can be readily observed following the mixing of asuitable bacterial suspension and an immunoglobulin preparation from ananimal previously immunized with that bacterium. Estimation of thestrength (titer) of a given antibody preparation is accomplished bydiluting the antibody (e.g. 2-fold serial dilutions) and determining thelast dilution which shows an agglutination effect. Visualization ofbacterial agglutination is made by examining the pattern of sedimentedbacteria on the bottom of a U-shaped plastic microtiter plate.Non-agglutinated bacteria sediment into a tight button whereasagglutinated bacteria are hindered from forming a button and sedimentinto a diffuse pattern on the bottom of the plastic well.

Test antibody dilutions were made in microtiter plates followed by theaddition of appropriately diluted bacteria (formalin fixed Helicobacterpylori or E Coli). Buffer containing methylene blue was then added tofacilitate easy reading of agglutination end points. The plates wereexamined after an eighteen (18) hour incubation at 4° C. Thereafter, theextent of agglutination was determined and the results expressed as thelowest serial dilution of antibody which exhibited agglutination asdefined above.

The results of this study are shown in Table 2. The data indicateclearly that antibodies raised against Helicobacter pylori and containedwithin BCW react specifically at high titers (i.e. dilutions) toHelicobacter pylori and do not react significantly with other viral andbacterial antigens. Likewise, similar immunoglobulin preparations fromnon-immunized is cows or cows immunized with unrelated bacterial orviral antigens do not react significantly with Helicobacter pylori.

                                      TABLE 2                                     __________________________________________________________________________    CHARACTERIZATION OF HELICOBACTER PYLORI (H. pylori) IMMUNE AND NON-           IMMUNE BOVINE LACTOIMMUNOGLOBULIN PREPARATIONS BY BACTERIAL                   AGGLUTINATION.sup.a                                                           ANTIBODY TEST SAMPLE  AGGLUTINATING ANTIBODY                                  COLOSTRUM                                                                             IMMUNIZING                                                                             IgGl TEST         SPECIFIC ACTIVITY                          REFERENCE                                                                             ANTIGEN  (mg/mL)                                                                            ANTIGEN                                                                             TITER/mL                                                                             (TITER/mgIgGl)                             __________________________________________________________________________    1       H. pylori                                                                              75.4 H. pylori                                                                           256,000                                                                              3,400                                      2       HRV.sup.b                                                                              52.4 H. pylori                                                                           2,560  49                                         3       E. coli  10   H. pylori                                                                           320    32                                         4       None     27.3 H. pylori                                                                           1,280  47                                         1       H. pylori                                                                              75.4 E. coli                                                                             640    8.5                                        2       HRV      52.4 E. coli                                                                             640    12                                         3       E. coli  10   E. coli                                                                             8,000  800                                        4       None     27.3 E. coli                                                                             160    5.9                                        __________________________________________________________________________     .sup.a H. pylori and E. coli bacteria were adjusted to 8 × 10.sup.9     cells/mL and added to twofold antibody dilutions in microtiter plates.        Methylene blue (0.01%) was added to the bacterial diluent (0.01 M             phosphate  buffered saline pH 7.0) to enhance visualization of                agglutinated bacteria.                                                        .sup.b Human rotavirus (HRV).                                            

The dominant antibody class found in colostrum is immunoglobulin type G(IgG) of the IGGI isotype subclass. These antibodies possess thephysical properties described in Table 3.

                  TABLE 3                                                         ______________________________________                                        BIOPHYSICAL CHARACTERIZATION OF ANTIBODIES                                    CONTAINED IN COLOSTRUM                                                        ______________________________________                                        Dominant Immunoglobulin Class:                                                                       IgGl                                                   Molecular Weight:      160,000 Daltons                                        Sedimentation Coefficient (S.sub.20,w):                                                              6.7 s                                                  Extinction Coefficient (E .sub.280 1% ):                                                             12.2                                                   Isoelectric Point:     5.5-6.8                                                Carbohydrate Content:  3%                                                     ______________________________________                                    

EXAMPLE 4 Preparation of Feed Material

Following assay, as described above, the BCW samples were added tocommercially available Similac infant formula (Ross Laboratories) toyield a standard concentration of 17 mg IgGl/mL. Following combinationthe samples were heat treated and an antibiotic mixture added to resultin the production of a material free of viable bacteria. This is arequirement for the gnotobiotic piglet model. The individualconcentrations of the antibiotics were selected so as to benoninhibitory for Helicobacter pylori growth only. All other bacteriagrowth was inhibited.

The control feed was Similac a containing the same type and amount ofantibiotic mixture.

EXAMPLE 5 Experimental Animals

A piglet litter of 13 animals was aseptically delivered and maintainedin germ free incubators according to standard procedures forestablishing germ free animals.

From this litter 11 animals were randomly divided into two experimentalgroups:

Group I: 5 Piglets fed control Similac (D nutrient only; and

Group II: 6 Piglets fed Similac a containing Helicobacter pyloriantibody.

Both experimental groups were orally infected with Helicobacter pyloriat 3 days of age with 2×10⁹ CFU/ML following pretreatment withCimetidine.

Proof of Helicobacter pylori infection was established by means of ELISAassays to detect Helicobacter pylori-specific porcine antibodies inserum samples from each piglet as illustrated in Table 4. The datacontained in this Table indicates that both groups of animals clearlydevelop significant antibody levels of all three immunoglobulin isotypesin their sera following infection as compared to preinfection sera.

                                      TABLE 4                                     __________________________________________________________________________    ISOTYPE SPECIFIC ELISA TESTING OF INFECTED AND CONTROL                        GERMFREE PIGLET SERA FOR ANTIBODY TO H. pylori                                                 MEAN OPTICAL DENSITY                                         PIGLET GROUP     ISOTYPE SPECIFIC SERUM ANTIBODY                              AND TIME SAMPLED IgG    IgM     IgA                                           __________________________________________________________________________      ALL PIGLETS    <0.01  <0.02   <0.03                                           BEFORE INFECTION                                                              IMMUNE COLOSTRUM FED                                                                         0.58   0.32    0.38                                            AFTER INFECTION                                                               CONTROL (SIMILAC) FED                                                                        0.77   0.38    0.35                                            AFTER INFECTION                                                             __________________________________________________________________________

EXAMPLE 6 Feeding Regimen

Ten days following Helicobacter pylori infection, piglets were fedeither Similac® only (Group I) or Similac® containing Helicobacterpylori specific antibodies (Group II) at a concentration of 17 mgIgGl/ml. The animals each received 30 mL of feed material three timeseach day. Following feeding, each animal received 150 mL of mildreplacer diet which did not contain antibodies. The feeding protocolcontinued for a twenty day period.

EXAMPLE 7 Post Protocol Assessment

At age 33 days, blood samples were drawn from each animal and theanimals were euthanized and necropsied. The blood samples were processedto serum and analyzed for the presence of porcine anti-Helicobacterpylori antibodies. Gastric epithelium samples (1-2cm²) from fivedifferent anatomic regions were taken at necropsy and subsequentlyevaluated for bacterial colonization and histologic evidence ofinfection. The biopsies were taken from the cardiac, fundic, pyloric,antrum and diverticulum regions of the stomach.

Biopsy samples were placed on selective agar plates containing theselected antibiotic mixture and streaked. The innoculated plates wereincubated in gas jars in a reduced oxygen atmosphere consisting of 5%O₂, 10% CO₂ and 80% N₂ at 37° C. which is a gas mixture whichselectively facilitates Helicobacter pylori growth. The plates wereexamined for bacterial growth after 5-8 days. Suspected Helicobacterpylori colonies were subcultured onto fresh medium. Gram stains wereperformed on both the bacterial growth as well as on the mucoid materialassociated with the biopsies. Identity of the bacteria was confirmedusing standard enzymatic (catalase, oxidase, urease) and antibioticsensitivity (Nalidixic acid and Cephalothin) assays. The profilesprovided by these assays allow for the accurate definition of the typeof bacteria being examined. This methodology is standard in the art. Theresults for these studies are summarized in Tables 5 and 6.

Data on the reisolation of viable bacteria from the various pigletstomach regions examined is shown in Table 5. A comparison was madebetween piglets fed the immune product as compared to those receivingnonimmune nutrient only.

In piglets fed non-immune nutrient, small bacterial colonies typical ofHelicobacter pylori developed on the agar plates in 84% of the 5 stomachepithelium biopsy sites assayed. In comparison, piglets fed nutrientcontaining specific anti-Helicobacter pylori antibodies, colonies wereobserved in only 37% of the biopsies. Viable Helicobacter pyloribacteria were isolated from 100% of the control piglets (i.e. thoseanimals receiving nonimmune nutrient) whereas viable Helicobacter pyloribacteria were isolated from only 50% of the piglets fed the immuneproduct. Identity of the bacteria as being Helicobacter pylori wasconfirmed using biochemical assays. The differences are significant atthe 95% confidence level using the one-sided Students "t" test or thenonparametric ranking approach (Wilcoxin test).

                                      TABLE 5                                     __________________________________________________________________________    REISOLATION OF HELICOBACTER pylori FROM GERMFREE PIGLETS.sup.a                PIGLET                                                                              FEEDING                                                                              H. pylori COLONY GROWTH.sup.c    PIGLETS WITH H. pylori          NUMBER                                                                              GROUP.sup.b                                                                         CARDIA                                                                              FUNDUS                                                                              PYLORUS                                                                             ANTRUM                                                                              DIVERTICULUM                                                                            POSITIVE BIOPSIES/TOTAL.sup.                                                  d                               __________________________________________________________________________    1     I     +     -     +     +     -         3/5                             2     I     +     +     +     +     +         5/5                             3     I     -     -     -     -     -         0/5                             4     I     -     -     -     -     -         0/5                             5     I     -     -     -     -     -         0/5                             6     I     +     +     -     +     -         3/5                                                                           11/30 = 37%                     7     NI    +     +     +     +     +         5/5                             8     NI    +     +     +     +     +         5/5                             9     NI    +     +     +     +     +         5/5                             10    NI    +     -     -     -     +         2/5                             11    NI    +     -     +     +     +         4/5                                                                           21/25 = 84%                     __________________________________________________________________________     .sup.a Each piglet challenged with H. pylori (10 days earlier) was fed        either H. pylori immune colostrum (1.5 g IgGl/90 mL/day in 3 feedings)        diluted in Similac alone (nonimmune group) for 20 days.                       .sup.b I = Piglets fed diluted immune colostrum. NI = Piglets fed             nonimmune preparation (Similac RTF).                                          .sup.c Based upon colony growth from selective agar plates. H. pylori         confirmed by appropriate biochemical testing (Urease, Catalase, Oxidase       and sensitivity to nalidixic acid and cephalothin).                           .sup.d Statistical evaluation (2 sample, 1sided T test). Immune fed group     significantly less than nonimmune group (p = 0.0298).                    

In addition to the above, Gram-stains were performed on the mucoidmaterial isolated with the tissue biopsies following five days ofincubation. (Helicobacter pylori is a Gram-negative bacterium).Specifically, a determination of the number of biopsy specimens whichwere positive for Gram-negative bacteria was made. This information issummarized in Table 6. These data confirm the information contained inTable 5 in that the incidence of Gram-negative bacteria found in pigletsfed the non-immune nutrient is 78% as compared to the 35% incidencefound in piglets fed nutrient containing specific anti-Helicobacterpylori antibodies. This difference is significant at the 90% confidencelevel.

                                      TABLE 6                                     __________________________________________________________________________    COMPARISON OF H. pylori CULTURE REISOLATION AND GRAM                          STAIN OF MUCUS                                                                            PIGLETS WITH                                                                             GRAM STAIN (MUCUS).sup.c                                     FEEDING                                                                              H. pylori POSITIVE                                                                      GNR POSITIVE/                                          PIGLETS                                                                             GROUP.sup.a                                                                         BIOPSIES.sup.b /TOTAL                                                                    TOTAL TESTED                                           __________________________________________________________________________    1     I     3/5        5/5                                                    2     I     5/5        1/1                                                    3     I     0/5        0/5                                                    4     I     0/5        1/5                                                    5     I     0/5        1/5                                                    6     I     3/5        0/2                                                                11/30 = 37%                                                                               8/23 = 35%                                            7     NI    5/5        4/5                                                    8     NI    5/5        3/4                                                    9     NI    5/5        4/5                                                    10    NI    2/5        3/4                                                    11    NI    4/5        4/5                                                                21/25 = 84%                                                                              18/23 = 78%                                            __________________________________________________________________________     I = Piglets fed diluted immune colostrum. NI = Piglets fed nonimmune          preparation (Similac RTF).                                                    .sup.b Based upon colony growth from selective agar plates. H. pylori         confirmed by appropriate biochemical testing (Urease, Catalase, Oxidase       and sensitivity to nalidixic acid and cephalothin).                           .sup.c Gram stain of inoculum mucus from plates with or without typcial H     pylori microcolonies (GNR = Gram Negative Rods).                         

Of critical importance to the interpretation of the data discussed aboveis the determination that all experimental animals had indeed beeninfected with Helicobacter pylori bacteria. Proof of this is indicatedby the data contained in Table 4 which illustrates that followinginfection with Helicobacter pylori all of the piglets developedantibodies to Helicobacter pylori (i.e. seroconvert) and containantibodies (immunoglobulins) within their sera exhibiting activityspecifically against Helicobacter pylori. This was determined by anELISA analysis of the sera of the piglets upon termination of theexperiment. The data contained in Table 4 indicates clearly that allanimals developed significant amounts of specific anti-Helicobacterpylori antibodies and therefore must have been infected with theHelicobacter pylori microorganism.

INDUSTRIAL APPLICABILITY

The presence of Helicobacter pylori in the gastrointestinal tract ofhumans is believed to cause gastritis. Previous therapies have seriousside effects and often fail to prevent reoccurance of the malady. Themedical community has long sought a therapy or preventative to thisdisorder and the present invention fills that need.

The foregoing detailed description is given for clearness ofunderstanding only, and no unnecessary limitation should be understoodtherefrom, as modifications will be obvious to those skilled in the art.

We claim:
 1. A composition of matter having utility for providingpassive immunity to a human when the composition is enterally ingested,said composition comprising non-denatured immunoglobulins whichspecifically bind to the bacterium Helicobacter pylori in the stomach ofa human, said Helicobacter pylori being capable of colonizing andproducing gastritis in humans and gnotobiotic piglets.
 2. A compositionof matter according to claim 1 wherein said immunoglobulins are isolatedfrom the mammary secretions of cows.
 3. A method for producing acomposition of matter having utility for providing passive immunity to ahuman when the composition is enterally ingested, said compositioncomprising immunoglobulins which specifically bind to the bacteriumHelicobacter pylori, said method comprising the steps of:(a) immunizinga lactating or pregnant cow with either a cell suspension or antigenderived from a strain of Helicobacter pylori capable of colonizing andproducing gastritis in humans or gnotobiotic piglets; (b) obtainingcolostrum or milk from the cow; and (c) isolating the immunoglobulinsfrom said colostrum or milk.